Xylitol dehydrogenase of acetic acid bacteria and gene thereof

ABSTRACT

Xylitol is produced by allowing xylitol dehydrogenase or cells instoduced with a DNA coding for xylitol dehydrogenase, which is a protein of the following (A) or (B) to act on D-xylulose, and collecting produced xylitol:
         (A) a protein which has the amino acid sequence of SEQ ID NO: 4;   (B) a protein which has the amino acid sequence of SEQ ID NO: 4 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has xylitol dehydrogenase activity.

CONTINUING APPLICATION INFORMATION

The present application is a continuation of application Ser. No.09/802,853, filed on Mar. 12, 2001, abandoned, which is a divisional ofapplication Ser. No. 09/363,189, filed on Jul. 29, 1999, now U.S. Pat.No. 6,242,228.

TECHNICAL FIELD

The present invention relates to a novel xylitol dehydrogenase of aceticacid bacteria, gene coding for the same, method for producing xylitoldehydrogenase, and method for producing xylitol. Xylitol is useful inthe fields of food industry, drug industry and the like.

BACKGROUND ART

Xylitol, which is a naturally occurring sugar alcohol, is a promisinglow-calorie sweetener because it has lower calories but exhibitscomparable sweetness compared with sucrose. In addition, because of itsanti-dental caries property, it can be a dental caries preventivesweetener. Furthermore, because xylitol does not elevate glucose level,it has been utilized for fluid therapy in the treatment of diabetesmellitus. For these reasons, it is expected that the demand of xylitolwill increase in future.

The current industrial production of xylitol mainly relies onhydrogenation of D-xylose as disclosed in U.S. Pat. No. 4,008,285.D-Xylose used as a raw material is obtained by hydrolysis of plantmaterials such as trees, straws, corn cobs, oat hulls and otherxylan-rich materials.

However, such D-xylose produced from hydrolysis of plant materialssuffers from a drawback that it is rather expensive, and it is arisenfrom high production cost. For example, the low yield of the hydrolysistreatment of plant materials leads to low purity of the producedD-xylitol. Therefore, the acid used for the hydrolysis and the dyes mustbe removed by ion exchange treatment after the hydrolysis treatment, andthe resulting D-xylose must be further crystallized to remove otherhemicellulose saccharides. In order to obtain D-xylose suitable forfoodstuffs, further purification would be required. Such ion exchangetreatment and crystallization treatment invite the increase ofproduction cost.

Therefore, several methods for producing xylitol have been developed,which utilize readily available raw materials and generate little waste.For example, there have been developed methods for producing xylitolutilizing other pentitols as a starting material. One of such readilyavailable pentitols is D-arabitol, and D-arabitol can be produced byusing yeast (Can. J. Microbiol., 31, 1985, 467-471; and J. Gen.Microbiol., 139, 1993, 1047-54).

Thus, several methods for producing xylitol that utilize D-arabitol as araw material have been developed. One method has been reported inApplied Microbiology, 18, 1969, 1031-1035, wherein D-arabitol isproduced from glucose by fermentation using Debaryomyces hanseniiATCC20121, then converted into D-xylulose using Acetobacter suboxydans,and the D-xylulose is converted into xylitol by the action of Candidaguilliermondii var. soya.

EP 403 392A and EP421 882A disclose methods which comprise producingD-arabitol by fermentation using an osmosis-resistant yeast, thenconverting D-arabitol into D-xylulose using a bacterium belonging to thegenus Acetobacter, Gluconobacter, or Klebsiellal, forming a mixture ofxylose and D-xylulose from the D-xylulose by the action of glucose(xylose) isomerase, and converting the produced mixture of xylose andD-xylulose into xylitol by hydrogenation. There is also disclosed theproduction of xylitol comprising preliminarily concentrating xylose inthe mixture of xylose and D-xylulose and converting the concentratedxylose into xylitol by hydrogenation.

While those methods for the production of xylitol utilizing D-arabitolas a starting material mentioned above can produce xylitol with arelatively high yield, however, they suffer from a drawback that theyrequires multiple reaction steps, and hence the processes should becomecomplicated. Therefore, they have not been economically acceptable.

On the other hand, breeding of xylitol fermenting microorganisms hasbeen attempted by using genetic manipulation techniques. InternationalPublication WO94/10325 discloses production of xylitol from glucosethrough fermentation by using a recombinant microorganism obtained byintroducing an arabitol dehydrogenase gene derived from a bacteriumbelonging to the genus Klebsiella and a xylitol dehydrogenase genederived from a bacterium belonging to the genus Pichiainto an arabitolfermenting microorganism (yeast belonging to the genus Candida,Torulopsis, or Zygosaccharomyces.

However, such breeding of xylitol fermenting microorganisms by usinggenetic manipulation techniques as mentioned above is not considered tobe completed as a practical means.

By the way, xylitol dehydrogenase is an enzyme that catalyzes thereaction producing xylitol from xylulose, and its presence has beenknown in various organisms. For example, there has been known thepresence of xylitol dehydrogenase in yeast species such as Pichiastipitis (J. Ferment. Bioeng., 67, 25 (1989)), Pachysolen tannophilus(J. Ferment. Technol., 64, 219 (1986)), Candida shehatae (Appl. Biochem.Biotech., 26, 197 (1990)), Candida parapsilosis (Biotechnol. Bioeng.,58, 440 (1998)), Debaryomyces hansenii (Appl. Biochem. Biotech., 56, 79(1996)), and Pullularia pullulans (An. Acad. Brasil. Cienc., 53, 183(1981)), filamentous bacteria such as Aspergillus niger (Microbiology,140, 1679 (1994)) and Neurospora crassa (FEMS Microbiol. Lett., 146, 79(1997)), algae such as Galdieria sulphuraria (Planta, 202, 487 (1997)),bacteria such as Morgannela morganii (J. Bacteriol., 162, 845 (1985)),and the like.

As for the xylitol dehydrogenase gene, there have been reportednucleotide sequences of the gene derived from Pichia stipitis (FEBSLett., 324, 9 (1993)) and Morgannela morganii (DDBJ/GenBank/EMBLaccession No. L34345).

However, xylitol dehydrogenase derived from acetic acid bacteria and itsgene have not been known so far even for their presence itself.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an enzyme involved inthe xylitol biosynthesis of microorganisms excellent in xylitolproduction ability, genes thereof, and use thereof in order to establisha technique for efficiently producing xylitol or breeding of xylitolfermenting bacteria.

To achieve the aforementioned object, the present inventors searchedmicroorganisms having ability to directly convert D-arabitol to xylitol.As a result, they found that certain bacteria belonging to the genusGluconobacter or Acetobacter have such ability. Further, they succeededin purifying two kinds of xylitol dehydrogenase from one of suchbacteria, Gluconobacter oxydans, and also succeeded in isolating genescoding for these enzymes and determining their structures. Thus, thepresent invention has been accomplished.

That is, the present invention provides:

-   (1) a protein defined in the following (A) or (B):

(A) a protein which has the amino acid sequence of SEQ ID NO: 4 inSequence Listing;

(B) a protein which has the amino acid sequence of SEQ ID NO: 4 inSequence Listing including substitution, deletion, insertion, addition,or inversion of one or several amino acids, and has xylitoldehydrogenase activity; and

-   (2) a protein defined in the following (C) or (D):

(C) a protein which has the amino acid sequence of SEQ ID NO: 6 inSequence Listing;

(D) a protein which has the amino acid sequence of SEQ ID NO: 6 inSequence Listing including substitution, deletion, insertion, addition,or inversion of one or several amino acids, and has xylitoldehydrogenase activity.

The present invention also provides:

-   (3) a DNA which codes for a protein defined in the following (A) or    (B):

(A) a protein which has the amino acid sequence of SEQ ID NO: 4 inSequence Listing;

(B) a protein which has the amino acid sequence of SEQ ID NO: 4 inSequence Listing including substitution, deletion, insertion, addition,or inversion of one or several amino acids, and has xylitoldehydrogenase activity;

-   (4) a DNA which codes for a protein defined in the following (C) or    (D):

(C) a protein which has the amino acid sequence of SEQ ID NO: 6 inSequence Listing;

(D) a protein which has the amino acid sequence of SEQ ID NO: 6 inSequence Listing including substitution, deletion, insertion, addition,or inversion of one or several amino acids, and has xylitoldehydrogenase activity;

-   (5) the DNA of the above item (3), which is a DNA defined in the    following (a) or (b):

(a) a DNA which contains at least a nucleotide sequence corresponding tonucleotide numbers 25 to 1053 of the nucleotide sequence of SEQ ID NO: 3in Sequence Listing;

(b) a DNA which is hybridizable with a DNA having a nucleotide sequencecorresponding to nucleotide numbers 25 to 1053 of the nucleotidesequence of SEQ ID NO: 3 in the Sequence Listing or a probe preparedfrom the nucleotide sequence under a stringent condition, and codes fora protein having xylitol dehydrogenase activity;

-   (6) The DNA of above item (5), the stringent condition is a    condition in which washing is performed at 60° C., and at a salt    concentration corresponding to 1×SSC and 0.1% SDS.-   (7) the DNA of the above item (4), which is a DNA defined in the    following (c) or (d):

(c) a DNA which contains at least a nucleotide sequence corresponding tonucleotide numbers 1063 to 1848 of the nucleotide sequence of SEQ ID NO:5 in Sequence Listing;

(d) a DNA which is hybridizable with a DNA having a nucleotide sequencecorresponding to nucleotide numbers 1063 to 1848 of the nucleotidesequence of SEQ ID NO: 5 in the Sequence Listing or a probe preparedfrom the nucleotide sequence under a stringent condition, and codes fora protein having xylitol dehydrogenase activity; and

-   (8) The DNA of above item (4), the stringent condition is a    condition in which washing is performed at 60° C., and at a salt    concentration corresponding to 1×SSC and 0.1% SDS.

The present invention also provides:

-   (9) a cell which is introduced with a DNA of any one of the above    items (3) to (8) in such a manner that xylitol dehydrogenase encoded    by the DNA can be expressed.

The present invention further provides:

-   (10) a method for producing xylitol dehydrogenase, which comprises    cultivating the cell of the above item (9) in a medium so that    xylitol dehydrogenase should be produced and accumulated in the    medium, and collecting xylitol dehydrogenase from the medium. The    present invention still further provides:-   (11) a method for producing xylitol, which comprises allowing    xylitol dehydrogenase of the above item (1) or (2) to act on    D-xylulose, and collecting produced xylitol; and-   (12) a method for producing xylitol, which comprises allowing the    cell of the above item (9) to act on D-xylulose, and collecting    produced xylitol.

While the xylitol dehydrogenase of the present invention has activityfor catalyzing both of the reaction for reducing D-xylulose to producexylitol, and the reaction for oxidizing xylitol to produce D-xylulose,the expression “having xylitol dehydrogenase activity” herein used meansto have at least the activity for catalyzing the reaction producingxylitol from D-xylulose.

According to the present invention, a novel xylitol dehydrogenase andDNA coding for the enzyme are provided, and xylitol dehydrogenase can beproduced by using the DNA.

Further, xylitol can be produced by using a cell introduced with thexylitol dehydrogenase or a DNA which codes for the enzyme.

Furthermore, the DNA of the present invention can be utilized for thebreeding of xylitol producing microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph for polyacrylamide gel electrophoresis ofpurified XDH; a) CBB staining after SDS-PAGE, b) CBB staining afterNative-PAGE, and c) activity staining after Native-PAGE.

FIG. 2 is a graph representing the pH dependency of the enzyme activityof XDH2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail hereafter.

<1> Xylitol Dehydrogenase of the Present Invention

The xylitol dehydrogenase of the present invention is an enzyme that isproduced by Gluconobacter oxydans. Two kinds of such enzyme were found,one was designated as XDH1, and the other as XDH2. XDH1 has the aminoacid sequence of SEQ ID NO: 4, and XDH2 has the amino acid sequence ofSEQ ID NO: 6 in Sequence Listing. XDH1 and XDH2 show a molecular weightof about 36,000 to about 40,000, and about 27,000 to about 30,000,respectively, as determined by SDS-PAGE (SDS polyacrylamide gelelectrophoresis). These two kinds of xylitol dehydrogenase, XDH1 and/orXDH2, may also be collectively referred to as “XDH” hereinafter.

As shown in Example 5 mentioned hereinafter, the optimum pH for XDH2 inthe reduction reaction (reaction producing xylitol from D-xylulose) wasaround 5. The optimum pH for the reduction reaction of well-knownxylitol dehydrogenases, for example, xylitol dehydrogenase derived fromAsperhillus niger is strictly 6.5 (Cor F. B. Witteveen, et al.,Microbiology, 140, 1679-1685, 1994), and therefore they are clearlydifferent from XDH2 of the present invention derived from Gluconobacterbacteria in the optimum reaction pH.

As an example of the method for producing XDH of the present invention,methods utilized for isolation and purification of XDH fromGluconobacter oxydans will be explained below.

First, cells of Gluconobacter oxydans, for example, the strain ATCC621,are disrupted by a mechanical means such as ultrasonication, or anenzymatic means utilizing a cell wall digesting enzyme etc., and a cellextract is prepared by removing the insoluble fraction therefrom bycentrifugation or the like.

The cell extract obtained as described above can be fractinated by acombination of conventional purification methods for proteins such asanion exchange chromatography, affinity chromatography, hydrophobicchromatography, and gel filtration chromatography, to purify XDH.

As a carrier for anion exchange chromatography, Q-Sepharose FF (producedby Pharmacia), Mono-Q (produced by Pharmacia) and the like can bementioned. The extract containing XDH is passed through a column filledwith such a carrier so that the enzyme should be adsorbed on the column,and, after washing the column, the enzyme is eluted with a buffer ofhigh salt concentration. In this case, the salt concentration may beraised stepwise, or a concentration gradient may be applied. Forexample, when Q-Sepharose FF is used, XDH adsorbed on the column may beeluted with 200 to 350 mM KCl. In the case of Mono-Q, it may be elutedwith 150 to 250 mM KCl.

As a carrier for affinity chromatography, HiTrap Blue (produced byPharmacia) can be mentioned. The XDH of the present invention utilizesNAD or NADH as a coenzyme, and hence has affinity for these substances.XDH adsorbed on the carrier can be eluted with a buffer containing about5 mM NAD.

As a carrier for hydrophobic chromatography, Phenyl Sepharose HP(produced by Pharmacia) can be mentioned. XDH adsorbed on the carrier ata low salt concentration can be eluted with about 200 to 300 mM ammoniumsulfate.

The XDH purified as described above can be further purified andseparated into XDH1 and XDH2 by gel filtration chromatography, SDS-PAGEor the like. As a carrier for gel filtration chromatography, Sephadex200HP (produced by Pharmacia) can be mentioned.

In the aforementioned purification procedure, if a fraction contains XDHor not can be confirmed by measuring the XDH activity of the fractionby, for example, the method shown in the examples mentioned hereinafter.

The N-terminus amino acid sequences of XDH1 and XDH2 purified asdescribed above are shown as SEQ ID NO: 1 and SEQ ID NO: 2 in SequenceListing, respectively.

While the XDH of the present invention can be obtained from cells ofGluconobacter oxydans by isolation and purification as described above,it can also be produced by introducing a DNA which codes for XDHmentioned hereinafter into a suitable host so that expression of the DNAshould be obtained in accordance with a conventionally used method forproducing heterogenous proteins by fermentation.

The various genetic recombination techniques mentioned below aredescribed in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

As a host for obtaining the expression of the XDH gene, variousprokaryotic cells including Escherichia bacteria such as Escherichiacoli, Gluconobacter bacteria such as Gluconobacter oxydans, and Bacillussubtilis, and various eukaryotic cells including Saccharomycescerevisiae, Pichia stipitis and Aspergillus oryzae can be used.

A recombinant DNA used for introducing the XDH gene into a host can beproduced by inserting a DNA coding for XDH into a vector selecteddepending on the kind of the host in which the expression is to beobtained in such a manner that the expression of XDH encoded by the DNAcan be possible. When an XDH gene specific promoter can function in thehost cell, that promoter can be used as the promoter for the expressionof the XDH gene. Further, if required, another promoter that canfunction in the host cell may be ligated to a DNA coding for XDH toobtain the expression under the control of that promoter. WhenEscherichia bacteria are used as a host, as examples of such a promoter,lac promoter, trp promoter, trc promoter, tac promoter, P_(R) promoter,P_(L) promoter of lambda phage and the like can be mentioned. As vectorsfor Escherichia bacteria, pUC19, pUC18, pBR322, pHSG299, pHSG298,pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 and the likecan be mentioned. Phage DNA vectors can also be utilized. Furthermore,an expression vector containing a promoter and capable of expressing aninserted DNA sequence can also be used.

E. coli can be transformed by introducing a plasmid in accordance with,for example, a method of D. A. Morrison (Methods in Enzymology, 68, 326(1979)) or a method in which recipient cells are treated with calciumchloride to increase permeability for DNA (Mandel, M. and Higa, A., J.Mol. Biol., 53, 159 (1970)).

<2> DNA Coding for XDH

A DNA coding for XDH can be obtained from a cDNA library or chromosomeDNA library of Gluconobacter oxydans by PCR (polymerase chain reaction,see White, T. J. et al; Trends Genet., 5, 185 (1989)) or hybridization.Primers used for PCR can be designed based on the amino acid sequencesof the amino termini determined for the purified XDH1 and XDH2. Further,since the nucleotide sequences of XDH1 gene (SEQ ID NO: 3) and XDH2 gene(SEQ ID NO: 5) have been elucidated according to the present invention,primers or probes for hybridization can be designed based on thosenucleotide sequences. By using primers having sequence corresponding to5′ non-translation region and 3′ non-translation region as primers forPCR, the XDH coding region can be amplified in its full length.Specifically, as for XDH2, a primer having a nucleotide sequence of aregion upstream from the nucleotide number 1063 in SEQ ID NO: 5 can beused as the 5′ primer, and a primer having a sequence complementary to anucleotide sequence of a region downstream from the nucleotide number1851 can be used as the 3′ primer. As for XDH1, a primer having anucleotide sequence of a region upstream from the nucleotide number 25in SEQ ID NO: 3 can be used as the 5′ primer.

Synthesis of the primers can be performed by an ordinary method such asa phosphoamidite method (see Tetrahedron Letters, 22, 1859 (1981)) byusing a commercially available DNA synthesizer (for example, DNASynthesizer Model 380B produced by Applied Biosystems). Further, the PCRcan be performed by using, for example, Gene Amp PCR System 9600produced by PERKIN ELMER and using TaKaRa LA PCR in vitro Cloning Kit(supplied by Takara Shuzo Co., Ltd.) in accordance with a methoddesignated by the suppliers.

The DNA of the present invention may code for XDH1 or XDH2 includingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids at one or a plurality of positions provided that theactivity to produce xylitol from D-xylulose of XDH1 or XDH2 encodedthereby is not deteriorated. Although the number of “several” aminoacids differs depending on the position or the type of amino acidresidues in the three-dimensional structure of the protein, it may be 2to 100, preferably 2 to 50, and more preferably 2 to 10.

DNA, which codes for the substantially same protein as XDH1 or XDH2 asdescribed above, is obtained, for example, by modifying the nucleotidesequence of XDH1 gene or XDH2 gene, for example, by means of thesite-directed mutagenesis method so that one or more amino acid residuesat a specified site of the gene involve substitution, deletion,insertion, addition, or inversion. DNA modified as described above maybe obtained by the conventionally known mutation treatment. The mutationtreatment includes a method for treating DNA coding for XDH in vitro,for example, with hydroxylamine, and a method for treating amicroorganism, for example, a bacterium belonging to the genusEscherichia harboring DNA coding for XDH with ultraviolet irradiation ora mutating agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) andnitrous acid usually used for the mutation treatment.

The substitution, deletion, insertion, addition, or inversion ofnucleotide as described above also includes mutation (mutant or variant)which naturally occurs, for example, the difference in strains, speciesor genera of the microorganism.

The DNA, which codes for substantially the same protein as XDH1 or XDH2,is obtained by expressing DNA having mutation as described above in anappropriate cell, and investigating the XDH1 or XDH2 activity of anexpressed product. The DNA, which codes for substantially the sameprotein as XDH1 or XDH2, is also obtained by isolating DNA which ishybridizable with DNA having, for example, a nucleotide sequencecorresponding to nucleotide numbers of 25 to 1053 of the nucleotidesequence of SEQ ID NO: 3 or a probe which can be prepared from the DNA,or a nucleotide sequence corresponding to nucleotide numbers of 1063 to1848 of the nucleotide sequence of SEQ ID NO: 5 or a probe which can beprepared from the DNA, under a stringent condition, and which codes fora protein having the XDH1 or XDH2 activity, from DNA coding for XDH1 orXDH2 having mutation or from a cell harboring it. The “stringentcondition” referred to herein is a condition under which so-calledspecific hybrid is formed, and non-specific hybrid is not formed. It isdifficult to clearly express this condition by using any numericalvalue. However, for example, the stringent condition includes acondition under which DNA's having high homology, for example, DNA'shaving homology of not less than 50% are hybridized with each other, andDNA's having homology lower than the above are not hybridized with eachother. Alternatively, the stringent condition is exemplified by acondition under which DNA's are hybridized with each other at a saltconcentration corresponding to an ordinary condition of washing inSouthern hybridization, i.e., 60° C., 1×SSC, 0.1% SDS, preferably0.1×SSC, 0.1% SDS.

The gene, which is hybridizable under the condition as described above,includes those having a stop codon generated in the gene, and thosehaving no activity due to mutation of active center. However, suchmutant genes can be easily removed by ligating the gene with acommercially available activity expression vector, and measuring theXDH1 or XDH2 activity in accordance with the method described below.

A DNA which codes for the XDH of the present invention can be used for,in addition to the method for producing XDH mentioned above and themethod for producing xylitol mentioned below, breeding of xylitolproducing microorganisms. For example, by enhancing the XDH gene in amicroorganism having the ability to convert D-arabitol into xylitol,e.g., Gluconobacter bacteria, the ability of the microorganism toconvert D-arabitol into xylitol can be increased.

<3> Method for Producing Xylitol

Xylitol can be produced by allowing the XDH of the present invention ora cell introduced with a DNA which codes for XDH and expresses XDH toact on D-xylulose, and collecting produced xylitol.

XDH may be an enzyme extracted from Gluconobacter bacteria, or an enzymeproduced by a genetic recombination technique utilizing a DNA whichcodes for XDH. Further, XDH may be either XDH1 or XDH2, and may be amixture of them at an arbitrary ratio.

The reaction producing xylitol from D-xylulose usually provides goodresults when performed at a temperature of 20-60° C., more preferably30-40° C., and pH of 4-10, more preferably pH of 4-8. For the reaction,either of standing culture or spinner culture may be used. While thereaction time may vary depending on concentration of XDH, amount ofcells, and substrate concentration to be used, it is desirably 1-100hours.

For collecting and separating the produced xylitol from a finishedreaction mixture, any conventional methods including use of a syntheticadsorbent, precipitant, or the like may be used.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further explained more specifically withreference to the following examples hereinafter. However, the presentinvention is not limited by the descriptions of the examples.

EXAMPLE 1 Production of XDH by Gluconobacter oxydans and PurificationThereof

<1> Culture of Gluconobacter oxydans ATCC621

The Gluconobacter oxydans strain ATCC621 was cultured to obtain itscells having sufficient XDH activity. The cultivation was alwaysperformed in PD medium as broth culture with shaking at 30° C. The PDmedium had a composition of 24 g/L potato dextrose (Difco), 30 g/L yeastextract (Difco), 5 g/L meat extract (Difco), 15 g/L glycerol, 10 g/LD-arabitol, 10 g/L D-xylose, 10 g/L xylitol, 20 g/L calcium carbonate(Kanto Chemical), pH 7.0.

First, as a seed culture, the strain ATCC621 was inoculated into aSakaguchi flask containing 40 ml of PD medium, and cultured overnightwith shaking at 30° C. The obtained culture broth was inoculated in anamount corresponding to 1% into 40 Sakaguchi flasks each similarlycontaining 40 ml of PD medium, and cultured with shaking for 3 days at30° C. (main culture). After removing calcium carbonate bycentrifugation, the cells were collected by centrifugation. The cellsobtained as described above were used as a material for purification ofXDH.

The XDH activity was determined by the following enzyme activity assay.30 μl of enzyme solution was added to 570 μl of a reaction solutioncontaining 100 mM (final concentration) xylitol, 2 mM NAD, and 100 mMCAPS (pH 10.0) to perform the enzymatic reaction at 30° C., and theincrease of NADH produced as the reaction proceeded was determined bymeasuring the absorbance at 340 nm using a spectrophotometer (DU640Spectrometer produced by BECKMAN). The activity producing 1 μmol of NADHper minute was defined as 1 unit (U). The calculation was performed byusing the molecular absorption coefficient ε of NADH at 340 nm of6.3×10³.

<2> Purification of XDH

(1) Preparation of Cell Extract

The cells obtained above were suspended in 50 mM potassium phosphatebuffer (pH 7), and centrifuged at 5000×g for 10 minutes to be collectedagain in the precipitated fraction. The procedure comprising suspensionand centrifugation of these cells was repeated twice to wash the cells.

About 10 g of the washed cells were suspended in 50 ml of Buffer 1 (20mM Tris-HCl (pH 7.6), 0.5 mM EDTA, 1 mM MgCl₂, 1 mM DTT), and disruptedby sonication for 20 minutes at 4° C. The disrupted cell suspension wascentrifuged (8000 rpm, 10 minutes) to remove the cell debris, andfurther ultracentrifuged (56000 rpm, 30 minutes) to remove the insolublefraction. Thus, a soluble fraction was obtained.

(2) Anion Exchange Chromatography

The above-obtained soluble fraction was loaded on an anion exchangechromatography column Q-Sepharose FF (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier.

The protein not adsorbed on the carrier (non-adsorbed proteins) waswashed off by using Buffer 1, and then the adsorbed protein was elutedby using a buffer containing KCl as an eluate. In this elution, KClconcentration in the buffer was linearly changed from 0 M to 0.5 M. TheXDH activity was measured for each eluted fraction obtained by thiselution, and the XDH activity was found in eluted fractionscorresponding to the KCl concentration of about 200 to 350 mM.

(3) NAD Affinity Chromatography

The above-obtained fractions containing the XDH activity were combined,and dialyzed against Buffer 1. The solution after the dialysis wasfiltered through a 0.45 μm filter. The obtained filtrate was loaded onan NAD affinity column HiTrap Blue 5 ml (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier.

The protein not adsorbed on the carrier (non-adsorbed proteins) waswashed off by using Buffer 1, and then the adsorbed protein was elutedby using Buffer 2 (20 mM Tris-HCl (pH7.6), 0.5 mM EDTA, 1 mM MgCl₂, 1 mMDTT, 5 mM NAD) containing NAD as an eluate. As a result, XDH wasdetected in the eluted fractions.

(4) Anion Exchange Chromatography

The aforementioned eluted fractions containing XDH activity werefiltered through a 0.45 μm filter. The obtained filtrate was loaded onan anion exchange chromatography column Mono-Q (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier.

The protein not adsorbed on the carrier was washed off by using Buffer1, and then the adsorbed protein was eluted by using a buffer containingKCl as an eluate. This elution was performed by linearly changing KClconcentration in the buffer from 0 mM to 500 mM. The XDH activity wasmeasured for each eluted fraction obtained by this elution, and the XDHactivity was found in eluted fractions corresponding to the KClconcentration of about 150 to 250 mM.

(5) Hydrophobic Chromatography

The eluted fractions for which the activity was detected were dialyzedagainst Buffer 3 (50 mM potassium phosphate buffer, 1 M ammoniumsulfate, pH 7.0). The solution obtained after the dialysis was filteredthrough a 0.45 μm filter. The obtained filtrate was loaded on ahydrophobic chromatography column Phenyl Sepharose HP (produced byPharmacia) equilibrated with Buffer 3. By this operation, XDH wasadsorbed on the carrier.

The protein not adsorbed on the carrier was washed off by using Buffer3, and then the adsorbed protein was eluted by using Buffer 4 (50 mMpotassium phosphate buffer, pH 7.0) as an eluate. For this elution,ammonium sulfate concentration in the buffer was linearly changed from 1M to 0 M. The XDH activity was measured for each eluted fractionobtained by this elution, and the XDH activity was found in elutedfractions corresponding to the ammonium sulfate concentration of about200 to 300 mM.

(6) Analysis of Purified Fraction

The XDH-active fraction obtained by the aforementioned purification wassubjected to SDS-PAGE and stained with Coomassie Brilliant Blue. As aresult, it was confirmed that XDH had been purified to such a level thatXDH could be detected as two bands, and their molecular weights wereestimated to be about 27,000 to abut 30,000, and about 37,000 to about40,000, respectively (see FIG. 1). Henceforth, the protein correspondingto the band of molecular weight of about 36,000 to abut 40,000 isreferred to as XDH1, and the protein corresponding to the band ofmolecular weight of about 27,000 to about 30,000 as XDH2.

Further, the obtained active fraction was subjected to Native-PAGE(non-denaturation PAGE), and stained with Coomassie Brilliant Blue. As aresult, two bands corresponding to molecular weights of more than 100kDa were confirmed. When the gel after Native-PAGE was subjected toactivity staining with an activity staining solution (25 mM glycinebuffer, 2.5 mM NAD, 50 mM xylitol, 0.2 nM phenazine methosulfate, 0.2 mMtetranitro blue tetrazolium chloride), the XDH activity was detected inboth of the two corresponding bands, and it was confirmed that both ofthe proteins corresponding to the two bands detected in the SDS-PAGE hadXDH activity (FIG. 1). The purified fraction containing these XDH1 andXDH2 will sometimes be referred to simply as XDH hereinafter.

The increase of the XDH specific activity as a result of theaforementioned purification was determined. The XDH activity of theaforementioned cell extract and the active fraction obtained by thepurification was measured. As a result, it was found that the specificactivity per unit protein weight was increased by about 550 times by theseries of purification procedures. By the activity assay method used forthis measurement, the specific activity of the purified XDH wasestimated to be about 130 U/mg (30° C., pH 10).

(7) Determination of Amino Acid Sequence at Amino Terminus of XDH

The N-terminus sequence of the XDH purified as described above wassequenced as follows. That is, about 10 μg in terms of protein of thepurified XDH fraction was electrophoresed in polyacrylamide gel in thepresence of SDS, and then the XDH in the gel was blotted to a membranefilter, and analyzed for the amino acid sequence from the N-terminus bya protein sequencer. Specifically, the objective enzymes were blotted toa polyvinylidene fluoride (PVDF) membrane from the gel after theelectrophoresis by the semi-dry method (Tanpakushitu Kozo Kaiseki[Analysis of Protein Structure], H. Hirano, Tokyo Kagaku Dojin) by usingMilliblot (Millipore). Then, the objective enzymes (XDH1 and XDH2) onthe PVDF film were analyzed by a protein sequencer (Model 476A producedby ABI) to perform N-terminus amino acid sequence analysis.

As a result, the amino acid sequence of 27 residues from the N-terminuswas determined for XDH1, and the amino acid sequence of 25 residues fromthe N-terminus was determined for XDH2. The amino acid sequence of thedetermined N-terminus sequence of XDH1 was shown as SEQ ID NO: 1 inSequence Listing, and the amino acid sequence of the N-terminus sequenceof XDH2 was shown as SEQ ID NO: 2 in Sequence Listing, respectively.

EXAMPLE 2 Conversion of D-xylulose into Xylitol by XDH

D-Xylulose was converted into xylitol using the purified XDH (XDH1 andXDH2) obtained in Example 1. 0.2 U of the purified XDH was added to 0.25ml of a reaction solution containing 21 mM D-xylulose, 20 mM NADH, and100 mM Tris-HCl buffer (pH 8.0), and incubated at 30° C. for 1 hour toallow the reaction. The solution after the reaction was subjected tohigh performance liquid chromatography (HPLC) to analyze the producedxylitol under the following conditions.

-   Column: Shodex SC1211 (produced by Showa Denko Co., Ltd.)-   Mobile phase: 50% acetonitrile/50% 50 ppm aqueous Ca-EDTA-   Flow rate: 0.8 ml/minute-   Temperature: 60° C.-   Detection: RI detector

As a result, formation of 18 mM xylitol was observed in the solutionafter the reaction, and it was shown that xylitol could be produced fromD-xylulose using the purified XDH.

EXAMPLE 3 Isolation of XDH Gene Derived from Gluconobacter

<1> Amplification of XDH Gene Fragment by PCR

-   (1) Preparation of PCR Primers Based on N-terminus Amino Acid    Sequence of XDH

Based on each of the aforementioned N-terminus amino acid sequences (SEQID NOS: 1 and 2) of XDH (XDH1, XDH2) derived from Gluconobacter oxydansATCC621, mixed primers which had the nucleotide sequences shown as SEQID NO: 7-10, respectively, were prepared.

-   (2) Preparation of Chromosome DNA of Gluconobacter oxydans ATCC621

Gluconobacter oxydans ATCC621 strain was cultured under the followingconditions. First, the ATCC621 strain was cultured in 20 ml of YPGmedium (3% glucose, 0.5% Bacto yeast extract, 0.3% Bacto peptone, pH6.5) overnight as a seed culture. By using 5 ml of this culture as seedbacteria, main culture was performed using 100 ml of YPG medium. Theculture was performed with shaking at 30° C.

After the bacteria were cultured to late log phase under theaforementioned conditions, 100 ml of the culture broth was centrifuged(12000×g, 4° C., 15 minutes) to collect the cells. The cells weresuspended in 10 ml of 50:20 TE (50 mM Tris-HCl, pH 8.0, 20 mM EDTA), andthe cells were washed and recovered by centrifugation. The cells weresuspended in 10 ml of 50:20 TE again. To this suspension, 0.5 ml of 20mg/ml lysozyme solution and 1 ml of 10% SDS solution were added, andincubated at 55° C. for 20 minutes. After the incubation,deproteinization was performed by adding equal volume of 10:1TE-saturated phenol. DNA was precipitated by adding equal volume of2-propanol to the separated aqueous layer, and collected. Theprecipitated DNA was dissolved in 0.5 ml of 50:20 TE, added with 5 μl of10 mg/ml RNase and 5 μl of 10 mg/ml Proteinase K, and allowed to reactat 55° C. for 2 hours. After the reaction, deproteinization wasperformed by adding equal volume of 10:1 TE-saturated phenol. Theseparated aqueous layer was further added with equal volume of 24:1chloroform/isoamyl alcohol, and stirred, and the aqueous layer wascollected. After this procedure was further repeated twice, the obtainedaqueous layer was added with 3 M sodium acetate solution (pH 5.2) sothat a final concentration of 0.4 M should be obtained, and furtheradded with twice as much volume of ethanol. The produced DNA wascollected as precipitates, washed with 70% ethanol, dried, and dissolvedin 1 ml of 10:1 TE.

-   (3) Preparation of DNA Fragment by PCR

The DNA molecule containing the gene coding for XDH derived fromGluconobacter bacteria was amplified and isolated by PCR using TaKaRa LAPCR in vitro Cloning Kit (supplied by Takara Shuzo Co., Ltd.). Theexperiments were performed according to the instruction attached to thekit hereafter unless otherwise indicated.

Five μg of the chromosome DNA produced as described in the above (2) wasdigested with restriction enzymes PstI or HindIII respectively. Then, aPstI cassette or HindIII cassette was ligated to the DNA fragmentscollected by the ethanol precipitation. Furthermore, after performingethanol precipitation, first PCR was performed for the collected DNA byusing a combination of primers of the primer C1 and primers mentionedbelow. That is, a DNA ligated to PstI cassette was used as a templateDNA for the primer XDH1-S1 that was based on the amino acid sequence ofXDH1, and a DNA ligated to HindIII cassette was used as a template DNAfor the primer XDH2-S1 that was based on the sequence of XDH2,respectively. There are shown the nucleotide sequences of the primer C1,the primer XDH1-S1, and the primer XDH2-S1 as SEQ ID NO: 11, SEQ ID NO:7, and SEQ ID NO: 9 in Sequence Listing, respectively. The primer C1 wascontained in the TaKaRa LA PCR in vitro Cloning Kit, and corresponded tothe sequence in the PstI cassette and the HindIII cassette. The PCRreaction was performed by using Gene Amp PCR System 9600 (produced byPERKIN ELMER), and a reaction according to the following conditions wasrepeated for 30 cycles.

94° C. for 30 seconds,

55° C. for 2 minutes

72° C. for 1 minute

Then, the reaction mixture was diluted 100 times, and newly added withthe primer C2 and the primer XDH1-S2 or the primer XDH2-S2, and thesecond PCR was performed. The conditions were the same as those of thefirst PCR. The nucleotide sequences of the primer C2, the primerXDH1-S2, and the primer XDH2-S2 are shown as SEQ ID NO: 12, SEQ ID NO:8, and SEQ ID NO: 10 in Sequence Listing, respectively. The primer C2was contained in the TaKaRa LA PCR in vitro Cloning Kit, and had thesequence corresponding to the sequence in the PstI cassette and theHindIII cassette. The primer XDH1-S2 and the primer XDH2-S2 eachcontained a sequence designed based on the amino acid sequencesdetermined, the sequence corresponding to EcoRI site, and EcoRI site.

After the reaction, 3 μl of the reaction mixture was subjected to 0.8%agarose gel electrophoresis. As a result, it was confirmed that a DNAfragment of about 1 kb was amplified when primer XDH1-S2 was used, and aDNA fragment of about 1.7 kb was amplified when XDH2-S2 was used.

-   (4) Cloning of DNA Fragments Amplified by PCR into pUC19

Cloning was performed by ligating the DNA fragments of about 1 kbp(XDH1) and about 1.7 kbp (XDH2) amplified by PCR with pUC19. Theligation was performed by using DNA Ligation Kit Ver.2 (supplied byTakara Shuzo Co., Ltd.). The experiments were performed according to theinstruction attached to the kit hereafter unless otherwise indicated.

400 ng of the DNA fragment of about 1 kb, which had been amplified byusing the primer XDH1-S2, was digested with PstI and EcoRI, thenpurified, and ligated to pUC19 digested with PstI and EcoRI. Escherichiacoli JM109 was transformed by using this ligation reaction mixture.

Further, 400 ng of the DNA fragment of about 1.7 kb, which had beenamplified by using the primer XDH2-S2, was digested with HindIII andEcoRI, then purified, and ligated to pUC19 digested with HindIII andEcoRI. Escherichia coli JM109 was transformed by using this ligationreaction mixture.

From the obtained transformant cells, several JM109 strains transformedwith pUC19 and containing the target DNA fragment of about 1 kbp (XDH1)or about 1.7 kbp (XDH2) were selected for each case. The selection wasperformed according to the method described in Molecular Cloning, 2ndedition, Cold Spring Harbor Press (1989).

-   (5) Determination of Nucleotide Sequence of XDH2 Gene Fragment

The plasmid carried by JM109 transformed with pUC19 containing the DNAfragment of about 1.7 kbp (XDH2) was prepared according to the methoddescribed in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989), and the nucleotide sequence of the inserted DNA fragment wasdetermined. The sequencing reaction was performed by using DyeTerminator Cycle Sequencing Kit (produced by ABI) according to theinstruction attached to the kit. The electrophoresis was performed byusing DNA Sequencer 373 (produced by ABI).

As a result, it was found that the DNA fragment amplified by PCR had asequence of from the thymidine residue at position 1116 to the thymidineresidue at position 2774 of the nucleotide sequence shown as SEQ ID NO:5 in Sequence Listing.

-   (6) Preparation of DNA Fragment of Upstream Region of XDH2 Gene by    PCR

The XDH2 gene and a DNA fragment of the upstream region of the XDH2 genewere amplified and isolated by PCR using the nucleotide sequencesdetermined above. The PCR reaction was performed by using TaKaRa LA PCRin vitro Cloning Kit (supplied by Takara Shuzo Co., Ltd.). Theexperiments were performed according to the instruction attached to thekit hereafter unless otherwise indicated.

Five μg of the chromosome DNA prepared as in the above (2) was digestedwith restriction enzyme SalI. Then, SalI cassette was ligated to the DNAfragment collected by ethanol precipitation. Ethanol precipitation wasfurther performed, and first PCR was performed for the collected DNA byusing the primer C1 and the primer XDH2UP-S1. The nucleotide sequencesof the primer C1 and the primer XDH2UP-S1 are shown as SEQ ID NO: 11 andSEQ ID NO: 13 in Sequence Listing, respectively. The primer XDH2UP-S1 isa sequence complementary to the region of from the cytosine residue atposition 1317 to the cytosine residue at position 1283 of the nucleotidesequence of the gene cluster coding for XDH2 of Gluconobacter shown asSEQ ID NO: 5.

The PCR reaction was performed by using Gene Amp PCR System 9600(produced by PERKIN ELMER), and a reaction according to the followingconditions was repeated for 30 cycles.

94° C. for 30 seconds,

55° C. for 2 minutes

72° C. for 1 minute

Then, the reaction mixture was diluted 100 times, and newly added withthe primer C2 and the primer XDH2UP-S2 to perform the second PCR. Theconditions were the same as those of the first PCR. The sequences of theprimer C2 and the primer XDH2UP-S2 are shown as SEQ ID NO: 12 and SEQ IDNO: 14 in Sequence Listing, respectively. The primer XDH2UP-S2 iscomposed of a sequence complementary to the region of from the guanosineresidue at position 1255 to the guanosine residue at position 1225 ofthe nucleotide sequence of the gene coding for XDH2 of Gluconobactershown as SEQ ID NO: 5. After the reaction, 3 μl of the reaction mixtureswas subjected to 0.8% agarose gel electrophoresis. As a result, it wasconfirmed that a DNA fragment of about 1.3 kb had been amplified.

-   (7) Determination of Nucleotide Sequence of XDH2 Gene and DNA    Fragment Containing Upstream Region Thereof

The DNA fragment of about 1.3 kbp amplified by the aforementioned PCRwas purified, and determined for the nucleotide sequence. The sequencingreaction was performed by using Dye Terminator Cycle Sequencing Kit(produced by ABI) according to the instruction attached to the kit. Theelectrophoresis was performed by using DNA Sequencer 373 (produced byABI).

As a result, it was found that the DNA fragment amplified in the above(6) had a sequence from the guanosine residue at position 1 to theguanosine residue at position 1224 of the nucleotide sequence shown asSEQ ID NO: 5 in Sequence Listing. The nucleotide sequence shown in SEQID NO: 5 comprises this nucleotide sequence combined with the nucleotidesequence determined in the above (5). The amino acid sequence which maybe encoded by this nucleotide sequence, deduced based on the universalcodons, is shown together in SEQ ID NO: 5, and also shown as SEQ ID NO:6. The sequence of from 2nd to 26th amino acid residues of that aminoacid sequence completely corresponded to the sequence of the 1st to the25th amino acid residues of the N-terminus amino acid sequence of XDH2shown as SEQ ID NO: 2. From this, it was confirmed that the DNAfragments amplified by the PCR were the target XDH2 gene and itsupstream region derived from Gluconobacter bacteria.

-   (8) Cloning of DNA Fragment Containing Full Length XDH2 Gene Coding    Region

Cloning was performed by amplifying a DNA fragment containing fulllength XDH2 gene coding region by PCR, and ligating it to pUC18. The PCRreaction was performed by using TaKaRa LAPCR kit (supplied by TakaraShuzo Co., Ltd.). The experiments were performed according to theinstruction attached to the kit hereafter unless otherwise indicated.

PCR was performed by using 1 μg of chromosome DNA of Gluconobacteroxydans ATCC621 strain produced in the same manner as in the above (2)as template, and using a primer XDH2-5′ and a primer XDH2-3′. Thenucleotide sequence of the primer XDH2-5′ was shown as SEQ ID NO: 15,and the nucleotide sequence of the primer XDH2-3′ was shown as SEQ IDNO: 16 in Sequence Listing. The primer XDH2-5′ comprises a sequencecorresponding to the region from the cytosine residue at position 1043to the adenosine residue at position 1063 of the nucleotide sequencecontaining the XDH2 gene shown as SEQ ID NO: 5, and the primer XDH2-3′comprises a sequence complementary to the region from the guanosineresidue at position 1957 to the cytosine residue at position 1978 of thesame.

The PCR reaction was performed by using GeneAmp PCR System 9600(produced by PERKIN ELMER), and a reaction according to the followingconditions was repeated for 30 cycles.

94° C. for 30 seconds,

55° C. for 2 minutes

72° C. for 1 minute

After the reaction, 3 μl of the reaction mixture was subjected to 0.8%agarose gel electrophoresis. As a result, it was confirmed that a DNAfragment of about 1 kbp had been amplified.

The DNA fragment of about 1 kbp amplified by the aforementioned PCR wasligated to pUC18 to perform cloning. The cloning was performed by usingDNA Ligation Kit Ver.2 (supplied by Takara Shuzo Co., Ltd.). Theexperiments were performed according to the instruction attached to thekit hereafter unless otherwise indicated. 400 ng of the amplified DNAfragment of about 1 kb was digested with BamHI and EcoRI, then purified,and ligated to pUC18 digested with BamHI and EcoRI. Escherichia coliJM109 was transformed by using this ligation reaction mixture.

From the obtained transformants, several JM109 strains transformed withpUC18 containing the target DNA fragment of about 1 kbp were selected.The selection was performed according to the method described inMolecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

The XDH2 gene derived form Gluconobacter oxydans could be cloned asdescribed above. The plasmid having the target XDH2 gene fragmentobtained by the aforementioned method is referred to as pUCXDH2.

-   (9) Determination of Nucleotide Sequence of XDH1 Gene Fragment

The plasmid carried by the JM109 strains transformed with pUC18containing the DNA fragment of about 1 kbp (XDH1) and selected above (4)was extracted according to the method described in Molecular Cloning,2nd edition, Cold Spring Harbor Press (1989), and the nucleotidesequence of the inserted DNA fragment was determined. The sequencingreaction was performed by using Dye Terminator Cycle Sequencing Kit(produced by ABI) according to the instruction attached to the kit. Theelectrophoresis was performed by using DNA Sequencer 373 (produced byABI).

As a result, it was found that the DNA fragment amplified by PCR had asequence from the guanosine residue at position 52 to the guanosineresidue at position 1011 of the nucleotide sequence shown as SEQ ID NO:3 in Sequence Listing.

-   (10) Cloning of XDH1 Gene from Chromosome DNA Library    i) Construction of Chromosome DNA Library

One μg of the chromosome DNA prepared in the above (2) was completelydigested with HindIII. After the DNA was collected by ethanolprecipitation, it was dissolved in 10 μl of 10:1 TE. Five μg of thissolution and 1 ng of pUC19 (supplied by Takara Shuzo Co., Ltd.) whichhad been digested with HindIII and subjected to dephosphorylation withBAP (bacterial alkaline phosphatase) were mixed, and the ligationreaction was performed by using DNA Ligation Kit Ver.2 (supplied byTakara Shuzo Co., Ltd.). Three μl of this ligation reaction mixture wasmixed with 100 μl of competent cells of Escherichia coli JM109 strain(supplied by Takara Shuzo Co., Ltd.) to transform the Escherichia coliJM109 strain. This was applied on a suitable solid medium to create achromosome DNA library.

ii) Preparation of Probe

It was decided to use a part of the XDH1 gene obtained in the above (3)for a probe. The DNA fragment of about 1 kb amplified by using theprimer C2 and the primer XDH1-S2, which was obtained in the above (3),was separated by 1% agarose gel electrophoresis. The target band wasexcised, and the DNA was purified by using Gene Clean II Kit (producedby Funakoshi). Finally, 16 μl of 50 ng/μl DNA solution was obtained. Aprobe labeled with digoxigenin was obtained by using this DNA fragmentand DIG High Prime (produced by Boehringer Mannheim) according to theinstruction attached to the product.

iii) Screening by Colony Hybridization

In order to obtain the XDH1 gene in full length, screening of thechromosome DNA library by colony hybridization utilizing theaforementioned probe was performed. The colony hybridization wasperformed according to the method described in Molecular Cloning, 2ndedition, Cold Spring Harbor Press (1989).

The colonies of the chromosome DNA library were blotted to a nylonmembrane filter (Hybond-N produced by Amersham), denatured with alkali,neutralized, and immobilized. Hybridization was performed by using EASYHYB (produced by Boehringer Mannheim). The filter was immersed into thebuffer (EASY HYB), and pre-hybridization was performed at 42° C. for onehour. Then, the labeled probe produced above was added, andhybridization was performed at 42° C. for 16 hours. Then, the filter waswashed with 2×SSC containing 0.1% SDS at room temperature for 20minutes. Further, it was washed twice with 0.1×SSC containing 0.1% SDSat 65° C. for 15 minutes.

The colonies hybridizable with the probe was detected by using DIGNucleotide Detection Kit (produced by Boehringer Mannheim) according tothe instruction attached to the kit. As a result, four strains ofcolonies hybridizable with the probe could be confirmed.

-   (11) DNA Sequence of XDH1 Gene

In the same manner as in the above (5), the nucleotide sequence of theDNA fragment inserted into pUC19 was determined. The result is shown asSEQ ID NO: 3 in Sequence Listing. The amino acid sequence, which isdeduced to be encoded by the nucleotide sequence based on universalcodons, is shown in SEQ ID NO: 3 together with the nucleotide sequence,and also shown as SEQ ID NO: 4. The amino acid sequence from the 2nd to28th amino acid residues completely corresponded to the sequencecomposed of the 27 residues of the 1st to the 27th amino acid residuesof the sequence shown as SEQ ID NO: 1. From this, it was confirmed thatthe obtained DNA fragment was the target XDH1 gene derived fromGluconobacter bacteria and flanking regions thereof.

EXAMPLE 4 Expression of XDH2 Gene Derived from Gluconobacter Bacteria inEscherichia coli and Purification of the Product

<1> Culture of Escherichia coli Harboring Recombinant XDH2 Gene andInduction of Expression

In the pUCXDH2 obtained in Example 3, the DNA coding for XDH2 genederived from Gluconobacter bacteria is ligated downstream of lacZpromoter, and therefore it was designed to be expressed under thecontrol of lacZ promoter.

Escherichia coli JM109 transformed with pUCXDH2, and Escherichia coliJM109 transformed with pUC18 as a control were cultured at 37° C.overnight with shaking in 50 ml of LB medium containing 100 μg/ml ofampicillin. These were used as seed culture. The seed culture ofEscherichia coli JM109 transformed with pUCXDH2 was inoculated in anamount of 1% to a flask containing fresh medium, and this was designatedas Experimental panel 1. On the other hand, the seed culture ofEscherichia coli JM109 transformed with pUC18 was similarly inoculatedin an amount of 1% to a flask, and this was designated as Experimentalpanel 2 (control). Each experimental panel was cultured, and whenabsorbance of the culture for a light having a wavelength of 610 nmbecame about 0.7, it was added with IPTG(isopropyl-beta-D-thiogalactopyranoside) to a final concentration of 1mM. Then, after 4 hours, the culture was completed.

<2> Confirmation of Protein Obtained by Induced Expression

After the completion of the cultivation, the cells were collected bycentrifugation (12,000×g, 15 minutes) of 10 ml of the culture broth. Thecells were suspended in 2 ml of 10 mM Tris-HCl, pH 7.5, washed andrecovered by centrifugation. The cells were suspended in 1 ml of thesame buffer, and disrupted by shaking with 0.1 mm zirconia beads for 3minutes using Multi Beads Shocker (Yasui Kikai). This disrupted cellsuspension was subjected to SDS-PAGE, and stained with CBB (CoomassieBrilliant Blue). As a result, a band corresponding to a molecular weightof about 27,000 to 30,000 was confirmed, which was observed only inExperimental panel 1 (JM109 transformed with pUCXDH2). Deduced from themolecular weight, it was considered that the desired XDH2 protein wasexpressed.

<3> Confirmation of XDH Activity

The XDH activity of the expressed protein was measured. The XDH activitywas measured by using the aforementioned disrupted cell suspensionaccording to the method described in Example 1. As a result, 14 U/mg ofthe XDH activity was detected in the Escherichia coli JM109 transformedwith pUCXDH2, whereas no XDH activity was detected in the Escherichiacoli JM109 transformed with pUC18 as the control. From this result, itwas confirmed that Escherichia coli JM109 transformed with pUCXDH2showed the XDH activity.

<4> Purification of XDH2 from Recombinant Escherichia coli JM109

The Escherichia coli JM109 cells transformed with pUCXDH2, which werecultured in the above <2>, were collected by centrifugation. Theobtained cells were used as a material for purification of XDH. The XDHactivity was measured by the method described in Example 1.

(1) Preparation of Cell Extract

The above bacterial cells were suspended in 50 mM potassium phosphatebuffer (pH 7), and collected again in a precipitated fraction obtainedby centrifugation at 5000×g for 10 minutes. This procedure comprisingsuspension and centrifugation was performed as washing of the cells.This washing process of the cells was repeated twice.

Three grams of the washed cells was suspended in 20 ml of Buffer 1 (20mM Tris-HCl (pH 7.6), 0.5 mM EDTA, 1 mM MgCl₂, 1 mM DTT), and disruptedby sonication for 20 minutes at 4° C. The disrupted suspension wascentrifuged (8000 rpm, 10 minutes) to remove cell residues, andultracentrifuged (56000 rpm, 30 minutes) to remove insoluble fraction.

(2) Anion Exchange Chromatography

The obtained soluble fraction was loaded on an anion exchangechromatography column Q-Sepharose FF (produced by Pharmacia)equilibrated by Buffer 1. By this operation, XDH was adsorbed on thecarrier.

The protein not adsorbed on the carrier (non-adsorbed protein) waswashed off by using Buffer 1, and then the adsorbed protein was elutedby using a buffer containing KCl as an eluate. In this elution, KClconcentration in the buffer was linearly changed from 0 M to 0.5 M. TheXDH activity was measured for each eluted fraction obtained by thiselution, and the XDH activity was found in eluted fractionscorresponding to the KCl concentration of about 200 to 350 mM.

(3) NAD Affinity Chromatography

The above-obtained fractions containing the XDH activity were combined,and dialyzed against Buffer 1. The solution after the dialysis wasfiltered through a 0.45 μm filter. The obtained filtrate was loaded onan NAD affinity column HiTrap Blue 5 ml (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier. Then, the protein not adsorbed on the carrier (non-adsorbedprotein) was washed off by using Buffer 1, and then the adsorbed proteinwas eluted by using, as an eluate, Buffer 2 (20 mM Tris-HCl (pH 7.6),0.5 mM EDTA, 1 mM MgCl₂, 1 mM DTT, 5 mM NAD) containing NAD. As aresult, XDH was detected in the eluted fractions.

(4) Hydrophobic Chromatography

The eluted fractions for which the activity was detected were dialyzedagainst Buffer 3 (50 mM potassium phosphate buffer, 1 M ammoniumsulfate, pH 7.0). The solution obtained after the dialysis was filteredthrough a 0.45 μm filter. The obtained filtrate was loaded on ahydrophobic chromatography column Phenyl Sepharose HP (produced byPharmacia) equilibrated with Buffer 3. By this operation, XDH wasadsorbed on the carrier.

Then, the protein not adsorbed on the carrier was washed off by usingBuffer 3, and then the adsorbed protein was eluted by using Buffer 4 (50mM potassium phosphate buffer, pH 7.0) as an eluate. For this elution,ammonium sulfate concentration in the buffer was linearly changed from 1M to 0 M. The XDH activity was measured for each eluted fractionobtained by this elution, and the XDH activity was found in elutedfractions corresponding to the ammonium sulfate concentration of about200 to 300 mM.

The obtained active fraction was subjected to SDS-PAGE, and stained withCoomassie Brilliant Blue. As a result, it was confirmed that XDH2 hadbeen purified to such a level that XDH2 could be detected as a singleband, and its molecular weight was estimated to be about 27,000 to abut30,000. That is, XDH2 expressed in Escherichia coli JM109 could bepurified as a single enzyme.

EXAMPLE 5 Determination of Optimum pH of XDH

By using the XDH2 enzyme obtained in Example 4, variation of the enzymeactivity depending on the reaction pH was measured as follows todetermine the optimum pH.

Sodium acetate buffers (pH 3.3, 4, 4.5, 5 and 6), Tris-HCl (pH 7 and 8),Glycine-NaOH (pH 9), and CAPS-NaOH (pH 10) buffers were used for theenzyme reaction buffers. Measurement of the XDH activity for thereduction reaction was performed as follows. Thirty μl of an enzymesolution was added to 570 μl of a reaction solution containing 100 mM(final concentration) D-xylulose, 0.2 mM NADH, and 100 mM buffer toallow the enzymatic reaction at 30° C., and the decrease of NADH causedby the reaction was determined by measuring the absorbance at 340 nmusing a spectrophotometer (DU 640 Spectrometer produced by BECKMAN). Theactivity decreasing 1 μmol of NADH per minute was defined as 1 U. Thecalculation was performed by using the molecular extinction coefficientε of NADH at 340 nm of 6.3×10³. Each buffer was added so that it shouldhave a concentration of 100 mM in the reaction solution. The XDHfraction purified above was used as an enzyme source, and the reactionwas performed at 30° C. The result of the measurement was represented asa relative value of enzyme activity to the actually determined pH valueof each reaction solution. For convenience, the activity for theoxidation reaction at pH 5 was defined as 100. The results of themeasurement are shown in FIG. 2.

It was found that the optimum pH for the reduction reaction (reactionproducing xylitol from D-xylulose) of the XDH2 of the present inventionwas about 5 (see FIG. 2). Since the optimum pH for the reductionreaction of the XDH derived from Asperhillus niger reported by Cor F. B.Witteveen, et al. (Microbiology, 140, 1679-1685, 1994) is strictly 6.5,and therefore the XDH2 of the present invention derived fromGluconobacter bacteria is clearly different from the known XDH in thereaction optimum pH. That is, it was demonstrated that, among theGluconobacter bacteria derived XDH found by the present invention, atleast XDH2 was characterized in that it had a lower optimum pH for thereduction reaction.

1. An isolated DNA defined in the following (c) or (d): (c) a DNAcomprising at least a nucleotide sequence corresponding to nucleotidenumbers 1063 to 1848 of the nucleotide sequence of SEQ ID NO: 5; (d) aDNA which contains at least a nucleotide sequence corresponding tonucleotide numbers 1063 to 1848 of the nucleotide sequence of SEQ ID NO:5 or a probe prepared from the nucleotide sequence under stringentconditions in which washing is performed at 60° C. and at a saltconcentration corresponding to 1×SSC and 0.1% SDS, and which codes for aprotein having xylitol dehydrogenase activity.
 2. The DNA of claim 1,which codes for (c).
 3. The DNA of claim 1, which codes for (d).
 4. Anisolated DNA which codes for a protein defined in the following (C) or(E): (C) a protein which has the amino acid sequence of SEQ ID NO: 6;(E) a protein which has the amino acid sequence of SEQ ID NO: 6including substitution, deletion, insertion or addition of 1 to 10 aminoacids, and has xylitol dehydrogenase activity.
 5. The DNA of claim 4,which codes for (E).
 6. A cell into which the DNA of claim 1 has beenintroduced.
 7. A cell into which the DNA of claim 2 has been introduced.8. A cell into which the DNA of claim 3 has been introduced.
 9. A cellinto which the DNA of claim 4 has been introduced.
 10. A cell into whichthe DNA of claim 5 has been introduced.
 11. A method for producingxylilol dehydrogenase, comprising cultivating the cell of claim 4 in asuitable medium for a time and under conditions suitable for productionof xylitol dehydrogenase.
 12. The method of claim 11, further comprisingcollecting the xylitol dehydrogenase from the culture medium.